303
Arab Univ.
QUANTITATIVE POLLEN ANALYSIS OF BEE HONEY AT CERTAIN
APIARIES IN QALYUBIA GOVERNORATE AND AVAILABLE HONEY
IN LOCAL MARKET, EGYPT
Mai, M. Hassanien; M.E. M. El- Sherif; A.A.A. Salem and M.A.M. Ali
Plant Protection Dept., Fac., of Agric., Ain Shams Univ., Cairo, Egypt
Keywords: Bee honey; Pollen analysis; Quantita-
tive pollen analysis
ABSTRACT
A new proposal method for quantitative pollen
analysis of pollen grains in each gram of bee hon-
ey was described, using a Neubauer improved
chamber for leucocytes and erythrocytes counting.
The study was conducted at the Bee Research
Unit, Dept. of plant protection, Faculty of Agricul-
ture, Ain Shams University, Shoubra El-kheimah,
Al Qalyubia, Egypt on 36 samples of citrus and
clover honeys. 18 samples were collected from 5
apiaries under study in Qalyubia Governorate;
Kanater, Shalakan, Tookh, Khankah and Shoubra
El kheimah and 18 samples were collected from
local market in 5 Governorates in Egypt; Cairo,
Giza, Qalyubia, Gharbia and Sharqia, during their
seasons of production in 2013, 2014 and 2015.
The results showed that 2.8 % of the samples
were poor in their content of pollen grains (2000-
10000 pollen grains /g honey), 77.8 % of the sam-
ples were rich in their content of pollen grains
(10000-50000 pollen grains /g honey), 11.1% of
the samples were more rich in their content of pol-
len grain (50000 -100000 pollen grains /g honey)
and 8.3% of the samples were very rich in their
content of pollen grain (>100000 pollen grains /g
honey).
INTRODUCTION
Bee honey contains numerous pollen grains
(mainly from plants bees feed on) of which provide
exact information for the quality of honey. Quanti-
tative pollen analysis is a branch of palynology
(Received 27 May, 2017)
(Revised 31 May, 2017)
(Accepted 4 June, 2017)
J. Agric. Sci.,
Ain Shams Univ.,
Cairo, 26(1), 303-311, 2018
[23]
(science of pollen and spores) and used in the
microscopic investigation of bee honey. The micro-
scopic analysis is a widely accepted assumption by
many scientists (Oddo and Piro, 2004). The
method provides essential information about the
hygienic aspects of bee honey production, its con-
tamination with mineral dust (Louveaux et al
1978). The approved quantitative pollen analysis
method was developed and proposed by the Inter-
national Commission for Bee Botany in 1978
(Louveaux et al 1978). The method was validated
in 2004 (von der Ohe et al 2004), and afterwards,
widely used in European laboratories for bee hon-
ey analysis. It is among the methods used for de-
scription of European honey types in the beginning
st
of the 21 century (Persano Oddo and Piro,
2004).
Haemocytometer (Neubauer improved cham-
ber) which used in routine laboratory practice for
leucocytes and erythrocytes counting (Heldrup et
al 1992), also used for counting pollen grains from
plants (Delaplane et al., 2013), and to evaluate the
number of pollen grains attached to bees (Human
et al 2013). Therefore, in the present work it was
used as a new method for quantitative pollen anal-
ysis of pollen grains in each gram honey.
In Egypt, most of researches were conducted
on chemical and physical properties [El- Sherbiny
et al (1980), Hassan (1985); Nour (1988); Gomaa
(2004); Rateb (2005); Faraj (2007) and Ismail
(2013)]. The accurate determination of bee honey
type especially pollen analysis requires more de-
tailed research. The proposed new method aims to
determine the number of pollen grains in each
gram of honey in all collected samples to evaluate
their quality.
304 Mai Hassanien; El-Sherif; Salem and Ali

MATERIALS AND METHODS the chamber to cover entirely the gridded areas
and to contact tightly the chamber edges Fig. (1).
36 samples of citrus and clover honeys were
collected from the 5 apiaries under study in
Qalyubia Governorate and local market from 5
Governorates of Egypt, during their seasons of
production in 2013, 2014 and 2015 as follows:
1- 9 citrus honey samples from certain apiaries in
Qalyubia Governorate situated in Kanater,
Shalakan, Tookh, Khankah and Shoubra El
kheimah included: H1, H2 and H3 in 2013,
H13, H14 and H27 in 2014 and H20, H21 and
H25 in 2015.
2- 9 clover honey samples from the same apiaries
included: H7, H8 and H9 in 2013, H17, H29
Fig.1. Neubauer improved chamber
and H32 in 2014 and H30, H31 and H33 in
2015.
Using a light microscope, eyepiece lens 10х
3- 9 citrus honey samples from the local markets
and objective lens 10x/0.24, the two grids of the
situated in Cairo, Giza, Qalyubia, Gharbia and
chamber were brought into focus and all pollen
Sharqia Governorates included: H4, H5 and H6
grains within them were counted using objective
in 2013, H15, H18 and H22 in 2014 and H19,
lens 40x/0.26, including those grains within each
H24 and H26 in 2015.
medium square and those that are over the top
4- 9 clover honey samples from the local markets
and right sides of the square (even when they are
situated in the same Governorates included:
partially out) Fig. (2).
H10, H11 and H12 in 2013, H16, H36 and H37
in 2014 and H28, H34 and H35 in 2015.
For quantitative analysis of pollen grains in
each gram of bee honey, 10 g of each honey sam-
ple were weighed in a graduated cylinder. Distilled
3
water was added to a total volume of 20 cm .The
obtained honey solution was divided into two ali-
3
quots of 10 cm in graduated centrifuge tubes. The
tubes were centrifuged at 2500×g for 10 min. With
3
an automated pipette, 9 cm of the supernatant
were discarded from each tube. To the remaining
3 3
amount of 1 cm in each of the two tubes, 5 cm
distilled water were added to a total volume of 6
3
cm . The two tubes were centrifuged at 2500×g for
3
10 min. With an automated pipette, 5 cm of the
supernatant were discarded from each tube. From
3
the remaining 1 cm honey solution, after a thor-
3 Fig. 2. Grid for pollen counting in Neubauer’s im-
ough mixing with a glass rod, 0.1 mm (μl) samples
proved cell counting chamber
were taken with automated pipettes from the bot-
tom of each tube. The amount was chosen due to
Pollen grains in both segments of the chamber
the experimentally established fact that a volume
were counted and the arithmetic mean (A) was
of .01 μl fills entirely, without leaking, the two semi-
calculated. The number of pollen grains in 10 g
reflective segments of the Neubauer improved
honey is calculated by the equation:
chamber after placing the cover glass.
Under direct light, the positions of the gridded
X = A × 50000
areas were identified and the 0.1 μl samples were
where:
pipetted in the middle of grids, resulting in two
Х – number of pollen grains in 10 g honey;
drops on each grid. A thin coverslip (Cover glass,
А – arithmetic mean of pollen grains counted in the
24 x 32 mm, thickness 0.13-0.17 mm), was then
two grids of the chamber (= (N1+ N2) /2).
carefully placed perpendicularly to the wide side of

Arab Univ. J. Agric. Sci., 26(1), 2018

 Quantitative pollen analysis of bee honey at certain apiaries in qalyubia
governorate and available honey in local market, Egypt
50000– coefficient for calculation of sample vol-
ume
For obtaining the coefficient 50000
Neubauer ’s improved chamber has 9 large
2
squares each with a 1 mm area, and each of them
3
could hold 0.1 mm of honey solution because
when putting the sample under the coverslip, the
cell suspension reaches a height of 0.1 mm
(Neubauer’s improved cell counting chamber,
2003). Taking these data into account, and con-
sidering one of the large squares, the volume will
be:
3 -4
1 x 1 x 0, 1 = 0, 1 mm = 10 ml
1. According to Hornitzky, 2009 the counted N
pollen grains in 5 of the medium squares (that
is, in 25 medium squares Fig. (3), the concen-
tration of our sample will be:
4
N x 10 pollen grain/ml
Fig. 3. Five medium squares (Hornitzky, 2009)
2. All the pollen grains within each medium
square (total 25 square) and those that are
over the top and right sides of the square (even
when they are partially out) are counted so the
4
final equation is: N * 5* 10 = N *50000
So, the number of pollen grains in each gram
honey is calculated by the equation:
X = A × 50000/10
where:
Х – number of pollen grains in each gram honey
А – arithmetic mean of pollen grains counted in the
two grids of the chamber (= (N1+ N2) /2).
5000– coefficient for calculation of sample volume
Arab Univ. J. Agric. Sci., 26(1), 2018
305
Example:
-
For obtaining the arithmetic mean (x ) of pollen
grains counted in the two grids of the chamber for
10 tests (A1-10) were as followed:
10 readings done in longer time intervals (1 month)
were made from each honey sample, the results
for pollen grains number in both chamber grids for
the ten tests (А1-10) were as followed:
А1 = 16 / 2 (number of chamber grids) = 8
А2 = 15 / 2 = 7.5
А3 = 15 / 2 = 7.5
А4 = 16 / 2 = 8
А5 = 15 / 2 = 7.5
A6 = 15 / 2 = 7.5
A7 = 16 / 2 = 8
A8 = 15 / 2 = 7.5
A9 = 16 /2 = 8
A10 =16 / 2 = 8
-
Х = 7.75
s. e. (standard error) =0.08
The calculated pollen grain numbers in 1 g
honey for each test run according to the formula
(Х1- 10) was as followed:
4
Х1 = 8 х 5000= 4*10 pollen grains/ 1 g honey
3
Х2 = 7, 5 х 5000 = 37.5*10 pollen grains / 1 g
3
Х3 = 7, 5 х 5000 = 37.5*10 pollen grains / 1 g
4
Х4 = 8 х 5000 = 4*10 pollen grains / 1 g
3
Х5 = 7, 5 х 5000 = 37.5*10 pollen grains / 1 g
3
Х6 = 7, 5 х 5000 = 37.5*10 pollen grains / 1 g
4
Х7 = 8 х 5000 = 4*10 pollen grains / 1g
3
Х8 = 7, 5 х 5000 = 37.5*10 pollen grains / 1 g
4
Х9 = 8 х 5000 = 4*10 pollen grains / 1 g
4
Х10 = 8 х 5000= 4*10 pollen grains / 1 g
-
Х = 387500
s. e. =4166.7
RESULTS AND DISCUSSION
The results from counting pollen grains in bee
honey samples collected from experimental apiar-
ies from Qalyubia Governorate and local market
in Egypt, produced in 2013, 2014 and 2015 are
presented in Tables 1 to 6.
The number of pollen grains of citrus honey
samples collected during 2013 from different apiar-
ies in Qalyubia Governorate and local market in
Egypt were summarized in Table (1). The number
of pollen grains in each gram of citrus honey col-
lected from Kanater (H1) significantly came the first
in order with an average of 365750 ± 16951.9 pol-
len grains / gram honey. Followed by the samples
collected from Tookh (H3), Shalakan (H2), Cairo
Univ. (H4), Ain Shams Univ. (H5) and Agri. Ministry
306 Mai Hassanien; El-Sherif; Salem and Ali
(H6) without any significant difference between
them and their averages were 26750 ±2814.9,
26200 ± 2493.5, 25750 ±2445.2, 17000 ±1658.3
and 14750 ± 2187.4 pollen grains / g honey, re-
spectively. Meanwhile, the number of pollen grains
of clover honey samples collected during the
same year 2013 from different apiaries in Qalyubia
Governorate and local market in Egypt were sum-
marized in Table (2). The number of pollen grains
in each gram of clover honey collected from Sha-
lakan (H8) significantly came the first in order and
its average was 47750 ± 2056.5 pollen grains / g
honey. Followed by the sample collected from
nd
Kanater (H7) which significantly came the 2 in
order and its average was 35750 ± 3051.6 pollen
grains / g honey. The samples collected from Cairo
Univ. (H11), Tookh (H9) and Agri. Ministry (H10)
rd
significantly came the 3 in order, without any sig-
nificant difference between them and their averag-
es were 21000 ±2114.8, 15250 ±1416.7 and 14250
±1750 pollen grains /g honey, respectively. While
the sample collected from Ain Shams Univ. (H12)
significantly came the last in order and its average
was 11000 ±1715.9 pollen grains / g honey without
any significant difference with the samples H9 and
H10.
The number of pollen grains of citrus honey
samples collected during 2014 from different apiar-
ies in Qalyubia Governorate and local market in
Egypt were summarized in Table (3). The number
of pollen grains in each gram of citrus honey col-
lected from Kanater (H27) significantly came the
first in order and its average was 88750 ± 2963.9
pollen grains / g honey. Followed by the sample
collected from Agri. Ministry (H18) which signifi-
nd
cantly came the 2 in order and its average was
54000 ± 1943.6 pollen grains / g honey. The sam-
ples collected from Ain Shams Univ. (H15) and
Gharbia Governorate (H22) significantly came the
rd
3 in order and their averages were 33500 ±
1870.8 and 31000 ±1632.9 pollen grains / g honey,
respectively. But the samples collected from Sha-
lakan (H13) and Tookh (H14) were the last in order
with averages of 18750 ± 1547.8 and 18750 ±
1070 pollen grains /g honey, respectively without
any significant difference between them. Mean-
while, the number of pollen grains of clover honey
samples collected during the same year 2014 from
different apiaries in Qalyubia Governorate and
local market in Egypt were summarized in Table
(4). The number of pollen grains in each gram of
clover honey collected from Shoubra El Khimah
(H32) and Shalakan (H29) significantly came the
first in order and their averages were 110250 ±
Arab Univ. J. Agric. Sci., 26(1), 2018
12469.1 and 109250 ± 5701.5 pollen grains / g
honey, respectively. Followed by the sample col-
lected from Agri. Ministry (H16) which significantly
nd
came the 2 in order and its average was 70500 ±
4561.3 pollen grains / g honey. While the samples
collected from Kanater (H17), Gharbia Gover-
norate (H37) and Ain Shams Univ. (H36) signifi-
cantly came the last in order and their averages
were 23750 ± 1193.2, 17500 ± 2075 and 12750 ±
870 pollen grains /g honey, respectively.
The number of pollen grains of citrus honey
samples collected during 2015 from different apiar-
ies in Qalyubia Governorate and local market in
Egypt were summarized in Table (5). The number
of pollen grains in each gram of citrus honey col-
lected from Gharbia Governorate, Tanta (H24)
significantly came the first in order and its average
was 49500 ± 1658.3 pollen grains /g honey. Fol-
lowed by the samples collected from Sharqia Gov-
ernorate (H26) and Shalakan (H21) which signifi-
nd
cantly came the 2 in order and their averages
were 40500 ± 2438.1 and 37250 ± 1204.7 pollen
grains /g honey, respectively. The samples collect-
ed from Kanater (H20) and Tookh (H25) signifi-
rd
cantly were the 3 in order and their averages
were 28750 ± 2334.8 and 22500 ± 1748 pollen
grains /g honey, respectively. But the sample col-
lected from Gharbia Governorate, Mahallah (H19)
significantly came the last in order and its average
was 20250 ± 946.5 pollen grains / g honey without
any significant difference with the sample H25.
Meanwhile, the number of pollen grains of clover
honey samples collected during the same year
2015 from different apiaries in Qalyubia Gover-
norate and local market in Egypt were summarized
in Table (6). The number of pollen grains in each
gram of clover honey collected from Gharbia Gov-
ernorate, Mahallah (H28) significantly came the
first in order and its average was 73000 ± 2549.5
pollen grains / g honey. Followed by the samples
collected from Shalakan (H31) and Kanater (H33)
nd
which significantly came the 2 in order and their
averages were 43250 ± 2008.7 and 43000 ± 3452
pollen grains / g honey. While the samples collect-
ed from Sharqia Governorate (H35) and Tookh
rd
(H30) significantly came the 3 in order and their
averages were 32750 ± 3060.7 and 26250 ±
1356.6 pollen grains / g honey. But the sample
collected from Gharbia Governorate, Tanta (H34)
was the last in order and its average was 8750 ±
1455.3 pollen grains / g honey.
 Quantitative pollen analysis of bee honey at certain apiaries in qalyubia 307
governorate and available honey in local market, Egypt

Table 1. Number of pollen grains / gram of Citrus honey samples collected from different locations at
Qalyubia Governorate and local market in 2013

Honey samp. Locations at Qalyubia Local market in Cairo

H4 H5 H6
H1 H2 H3
Cairo Ain Shams Agri.
Kanater Shalakan Tookh
Rep. Univ. Univ. Ministry

1 175004 25000 17500 30000 17500 5000

2 320000 15000 42500 15000 12500 15000
3 350000 32000 27500 20000 12500 7500
4 237500 20000 17500 15000 10000 10000
5 382500 20000 12500 22500 15000 7500
6 392500 40000 30000 30000 22500 17500
7 417500 35000 25000 32500 20000 22500
8 372500 20000 32500 22500 25000 20000
9 387500 30000 30000 35000 12500 17500
10 380000 25000 32500 35000 22500 25000
- a b b b b b
Mean(x ) 365750 26200 26750 25750 17000 14750
± ± ± ± ± ± ±
s. e. 16951.9 2493.5 2814.9 2445.2 1658.3 2187.4
L.S.D. 42617 pollen grains / g honey

Table 2. Number of pollen grains / gram of Clover honey samples collected from different locations at
Qalyubia Governorate and local market in 2013

Honey samp. Locations at Qalyubia Local market in Cairo

H12
H11 H10
H7 H8 H9 Ain
Cairo Agri.
Rep. Kanater Shalakan Tookh Shams
Univ. Ministry
Univ.
1 35000 52500 17500 20000 12500 15000
2 45000 55000 12500 20000 5000 12500
3 37500 50000 7500 20000 7500 10000
4 35000 57500 10000 30000 2500 10000
5 20000 45000 15000 15000 10000 12500
6 20000 42500 15000 25000 10000 10000
7 35000 50000 22500 17500 10000 10000
8 42500 37500 20000 10000 15000 27500
9 50000 40000 15000 20000 17500 17500
10 37500 47500 17500 32500 20000 17500
- b a cd c d cd
Mean(x ) 35750 47750 15250 21000 11000 14250
± ± ± ± ± ± ±
s. e. 3051.6 2056.5 1416.7 2114.8 1715.9 1750
L.S.D. 8702.4 pollen grains / g honey

Arab Univ. J. Agric. Sci., 26(1), 2018

308 Mai Hassanien; El-Sherif; Salem and Ali

Table 3. Number of pollen grains / gram of Citrus honey samples collected from different locations at
Qalyubia Governorate and local market in 2014

Honey samp. Locations at Qalyubia Local market in Gharbia and Cairo

H15 H18
H27 H13 H14 H22 Ain Agri.
Rep. Kanater Shalakan Tookh Gharbia Shams Ministry
Univ.
1 75000 10000 15000 35000 45000 47500
2 77500 17500 15000 25000 35000 47500
3 87500 22500 17500 25000 37500 52500
4 85000 20000 17500 35000 27500 55000
5 100000 27500 17500 27500 32500 65000
6 95000 22500 22500 25000 27500 60000
7 92500 15000 17500 32500 27500 60000
8 102500 15000 17500 32500 40000 50000
9 92500 17500 22500 32500 32500 47500
10 80000 20000 25000 40000 30000 55000
- a d d c c b
Mean(x ) 88750 18750 18750 31000 33500 54000
± ± ± ± ± ± ±
s. e. 2963.9 1547.8 1070.4 1632.9 1870.8 1943.6
L.S.D. 7374 pollen grains / g honey

Table 4. Number of pollen grains / gram of Clover honey samples collected from different locations at
Qalyubia Governorate and local market in 2014

Honey samp. Locations at Qalyubia Local market in Gharbia and Cairo

H36
H32 H16
H17 H29 H37 Ain
Rep. Shoubra Agri.
Kanater Shalakan Gharbia Shams
El-khimah Ministry
Univ.

1 20000 90000 77500 22500 10000 50000

2 25000 102500 112500 5000 17500 65000
3 25000 120000 90000 20000 50001 85000
4 27500 85000 70000 15000 10000 57500
5 17500 97500 137500 17500 10000 62500
6 20000 127500 95000 10000 12500 55000
7 30000 140000 185000 15000 10000 82500
8 25000 112500 165000 20000 12500 72500
9 22500 95000 80000 22500 15000 90000
10 25000 122500 90000 27500 15000 85000
- c a a c c b
Mean(x ) 23750 109250 110250 17500 12750 70500
± ± ± ± ± ± ±
s. e. 1193.2 5701.5 12469.1 2075 870 4561.3
L.S.D. 25028 pollen grains / g honey

Arab Univ. J. Agric. Sci., 26(1), 2018

 Quantitative pollen analysis of bee honey at certain apiaries in qalyubia 309
governorate and available honey in local market, Egypt

Table 5. Number of pollen grains / gram of Citrus honey samples collected from different locations at
Qalyubia Governorate and local market in 2015

Honey samp.
Locations at Qalyubia Local market Gharbia and Sharqeia

H19 H24
H20 H21 H25 H26
Gharbia Gharbia
Kanater Shalakan Tookh Sharqeia
Rep. (Mahallah) (Tanta)
1 20000 32500 27500 20000 52500 27500
2 27500 35000 22500 20000 42500 37500
3 37500 32500 15000 17500 50000 42500
4 27500 35000 15000 15000 60000 30000
5 37500 45000 20000 22500 45000 47500
6 27500 37500 20000 17500 55000 35000
7 37500 40000 32500 22500 47500 42500
8 32500 40000 27500 20000 45000 45000
9 17500 37500 22500 25000 50000 50000
10 22500 37500 22500 22500 47500 47500
- c b cd d a b
Mean(x ) 28750 37250 22500 20250 49500 40500
± ± ± ± ± ± ±
s. e. 2334.8 1204.7 1748 946.5 1658.3 2438.1
L.S.D. 7541.9 pollen grains / g honey

Table 6. Number of pollen grains / gram of Clover honey samples collected from different locations at
Qalyubia Governorate and local market in 2015

Honey samp. Local market in Gharbia and

Locations at Qalyubia
Sharqeia
H28 H34
H33 H31 H30 H35
Gharbia Gharbia
Rep. Kanater Shalakan Tookh Sharqeia
(Mahallah) (Tanta)

1 35000 52500 22500 75000 20000 40000

2 35000 35000 27500 67500 7500 50003
3 37500 47500 17500 90000 5000 55000
4 42500 32500 25000 75000 10000 27500
5 67500 47500 30000 62500 7500 37500
6 35000 42500 25000 62500 12500 22500
7 42500 40000 25000 77500 7500 25000
8 37500 50000 27500 70000 5000 30000
9 57500 42500 30000 75000 7500 25000
10 40000 42500 32500 75000 5000 30000
- b b C a d c
Mean(x ) 43000 43250 26250 73000 8750 32750
± ± ± ± ± ± ±
s. e. 3452 2008.7 1356.6 2549.5 1455.3 3060.7
L.S.D. 10203 pollen grains / g honey

Arab Univ. J. Agric. Sci., 26(1), 2018

310 Mai Hassanien; El-Sherif; Salem and Ali

As shown in Table (7) the honey samples (col-
lected during the 3 years 2103, 2014 and 2015)
were classified according to the categories of
Leaveaux, et al 1978, as follows:
 2.8% of the samples were poor in their content
of pollen grains (2000-10000 pollen grains/ g
honey).
 77.8% of the samples were rich in their con-
tent of pollen grains(10000-50000pollen
grains/ g honey).
 11.1% of the samples were more rich in their
content of pollen grains (50000-100000 pollen
grains/ g honey).
8.3% of the samples were very rich in their
content of pollen grains (>100000 pollen grains/ g
honey).
Table 7. Percentages of the 36 honey samples
(collected during the 3 years 2013, 2014 and 2015)
in each category of the Leaveaux, et al 1978 cat-
egories, according to their content of pollen grains
Categories of Percentages of
No. of
Leaveaux, et al honey
honey
1978 (pollen samples in
samples
grains/ g honey) each category
1. < 2000 0 0
2. 2000-10000 1 2.8
3. 10000-50000 28 77.8
4. 50000-100000 4 11.1
5. > 100000 3 8.3
Total 36 100
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